介體灰飛虱蛋白與水稻條紋病毒-NCP互作驗證及VAP-B在病毒傳播功能的初步分析
發(fā)布時間：2024-12-17 22:02
水稻生長發(fā)育過程中會受到多種病害的影響,其中條紋葉枯病在水稻產(chǎn)區(qū)多次間歇性暴發(fā),對水稻的生產(chǎn)構(gòu)成嚴重威脅。該病害常發(fā)生于溫帶和亞熱帶地區(qū),21世紀初在中國稻麥輪作區(qū)引起高達30%-40%的減產(chǎn)。水稻條紋葉枯病的病原是水稻條紋病毒(RSV),該病毒依靠介體灰飛虱(SBPH,Laodelphax striatellus)以持久增殖的方式水平傳播,并且還能經(jīng)過灰飛虱的卵進行垂直傳播�；绎w虱不僅可以取食水稻、小麥、燕麥、玉米和大麥等多種禾本科作物,同時也是RSV等重要植物病毒的傳播介體。RSV-SBPH的相互作用是一個復雜的機制,決定著病毒能否在自然界中成功傳播,并進一步影響病害的暴發(fā)流行。病毒在介體內(nèi)的侵染、運動和復制涉及到了大量的蛋白質(zhì)間相互作用。本研究利用酵母雙雜交技術(shù)明確了3種介體灰飛虱蛋白與RSV NCP之間的相互作用,這3種蛋白分別為灰飛虱糖轉(zhuǎn)運蛋白1、灰飛虱糖轉(zhuǎn)運蛋白2和囊泡相關(guān)膜蛋白相關(guān)蛋白B(VAP-B),并對這3種蛋白在介體灰飛虱內(nèi)的mRNA表達水平進行了檢測。同時進一步利用pull-down實驗證實了VAP-B與RSV NCP之間存在互作。隨后利用昆蟲的RNAi技術(shù)研究了V...
【文章頁數(shù)】：131 頁
【學位級別】：博士
【文章目錄】：
中文摘要
Abstract
CHAPTER-Ⅰ Literature review
1.1 Economic importance of rice
1.2 Rice stripe disease and its damage for rice
1.2.1 Symptomology and alternate host plants
1.3 Rice stripe virus
1.4 Significance of plant viruses transmitted by planthoppers
1.5 Laodelphax striatellus(SBPH):The vector of RSV
1.5.1 Biological characteristics
1.5.2 Distribution
1.5.3 Occurrence in China
1.6 Virus transmission mechanisms by insect vectors
1.6.1 Persistent propagative virus transmission
1.6.2 The transmission mechanism of tenuiviruses
1.6.3 Transmission route of rice stripe virus
1.7 A general overview of protein-protein interactions
1.7.1 Yeast two-hybrid system
1.7.2 A brief introduction to other protein-protein interaction verification methods
1.8 Purpose and significance of the research
CHAPTER-Ⅱ ST1,ST2 and VAP-B amplification and analysis
2.1 Materials and Methods
2.1.1 Total RNA Extraction
2.1.2 Reverse transcription and amplification of ST1,ST2 and VAP-B genes
2.1.3 Purification of PCR products
2.1.4 Ligation of purified DNA into the p MD-19T vector
2.1.5 Transformation of ligated product into E.coli
2.1.6 Plasmid extraction
2.1.7 Biological interpretation of ST1,ST2 and VAP-B genes
2.2 Results and analysis
2.2.1 Total RNA Extraction from SBPH
2.2.2 Amplification and Blast analysis of ST1,ST2 and VAP-B genes
2.2.3 Domain analysis and multiple sequence alignment of ST1,ST2 and VAP-B genes
2.2.4 Transmembrane prediction and phylogenetic analysis of ST1,ST2 and VAP-B genes
2.3 Discussion
CHAPTER-Ⅲ Verification of interaction between Ls ST1,Ls ST2 and VAP-B with RSV-NCP by Yeast two-hybrid
3.1 Materials and Methods
3.1.1 Yeast two-hybrid system to verify the interaction between RSV-NCP and Ls ST1,Ls ST2,and VAP-B
3.2 Results
3.2.1 Vector construction
3.2.2 Functional assay of Ls ST1,Ls ST2 and VAP-B(prey)with RSV-NCP(bait)in co-transfected yeast cells
3.3 Discussion
CHAPTER-Ⅳ mRNA expression level analysis by reverse transcription quantitative real-time PCR
4.1 Materials and Methods
4.1.1 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.2 Results
4.2.1 Primers constructed for RT-q PCR analysis
4.2.2 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.3 Discussion
5 CHAPTER-Ⅴ Validation of interaction between VAP-B with RSV-NCP by Pull-Down Assay
5.1 Materials and Methods
5.1.1 Pull-down assay to verify the interaction between VAP-B and RSV-NCP
5.2 Results
5.2.1 Vector construction
5.2.2 Pull-down assay
5.3 Discussion
CHAPTER-Ⅵ Effect of RNA interference of VAP-B on infection and relative expression of RSV in SBPH
6.1 Materials and Methods
6.1.1 ds RNA synthesis
6.1.2 Microinjection of the ds RNA to the SBPH
6.1.3 Effect of RNAi of VAP-B on infection and relative expression of RSV
6.1.4 Effect of RNAi of VAP-B on the transmission of RSV
6.2 Results
6.2.1 ds RNA construction
6.2.2 Effect of ds VAP-B injection on the expression of VAP-B in the SBPH
6.2.3 Effect of RNAi of VAP-B on infection and relative expression of RSV in the viruliferous SBPH
6.2.4 Effect of RNAi of VAP-B on the transmission of RSV
6.3 Discussion
CHAPTER-Ⅶ First reports of Tomato chlorosis virus and Potato leafroll virus in tomato from Pakistan
7.1 Materials and Methods
7.1.1 Samples collection
7.1.2 ELISA
7.1.3 RNA isolation and RT-PCR
7.1.4 Whitefly transmission
7.1.5 Next-Generation Sequencing
7.1.6 BLAST and phylogenetic analysis
7.1.7 Selection pressure and recombination analysis
7.2 Results
7.2.1 ELISA for different viruses
7.2.2 RT-PCR
7.2.3 Transmission test
7.2.4 Sequencing,BLAST and Phylogenetic analysis
7.2.5 Full genome sequence analysis
7.2.6 Selection pressure and recombination analysis
7.2.7 Gene flow and genetic differentiation analysis
7.2.8 Haplotype and nucleotide diversity
7.3 Discussion
CHAPTER-Ⅷ Summary
Bibliography
Acknowledgement
Author's Biography
Appendix Ⅰ
Appendix Ⅱ
本文編號：4016595
【文章頁數(shù)】：131 頁
【學位級別】：博士
【文章目錄】：
中文摘要
Abstract
CHAPTER-Ⅰ Literature review
1.1 Economic importance of rice
1.2 Rice stripe disease and its damage for rice
1.2.1 Symptomology and alternate host plants
1.3 Rice stripe virus
1.4 Significance of plant viruses transmitted by planthoppers
1.5 Laodelphax striatellus(SBPH):The vector of RSV
1.5.1 Biological characteristics
1.5.2 Distribution
1.5.3 Occurrence in China
1.6 Virus transmission mechanisms by insect vectors
1.6.1 Persistent propagative virus transmission
1.6.2 The transmission mechanism of tenuiviruses
1.6.3 Transmission route of rice stripe virus
1.7 A general overview of protein-protein interactions
1.7.1 Yeast two-hybrid system
1.7.2 A brief introduction to other protein-protein interaction verification methods
1.8 Purpose and significance of the research
CHAPTER-Ⅱ ST1,ST2 and VAP-B amplification and analysis
2.1 Materials and Methods
2.1.1 Total RNA Extraction
2.1.2 Reverse transcription and amplification of ST1,ST2 and VAP-B genes
2.1.3 Purification of PCR products
2.1.4 Ligation of purified DNA into the p MD-19T vector
2.1.5 Transformation of ligated product into E.coli
2.1.6 Plasmid extraction
2.1.7 Biological interpretation of ST1,ST2 and VAP-B genes
2.2 Results and analysis
2.2.1 Total RNA Extraction from SBPH
2.2.2 Amplification and Blast analysis of ST1,ST2 and VAP-B genes
2.2.3 Domain analysis and multiple sequence alignment of ST1,ST2 and VAP-B genes
2.2.4 Transmembrane prediction and phylogenetic analysis of ST1,ST2 and VAP-B genes
2.3 Discussion
CHAPTER-Ⅲ Verification of interaction between Ls ST1,Ls ST2 and VAP-B with RSV-NCP by Yeast two-hybrid
3.1 Materials and Methods
3.1.1 Yeast two-hybrid system to verify the interaction between RSV-NCP and Ls ST1,Ls ST2,and VAP-B
3.2 Results
3.2.1 Vector construction
3.2.2 Functional assay of Ls ST1,Ls ST2 and VAP-B(prey)with RSV-NCP(bait)in co-transfected yeast cells
3.3 Discussion
CHAPTER-Ⅳ mRNA expression level analysis by reverse transcription quantitative real-time PCR
4.1 Materials and Methods
4.1.1 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.2 Results
4.2.1 Primers constructed for RT-q PCR analysis
4.2.2 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.3 Discussion
5 CHAPTER-Ⅴ Validation of interaction between VAP-B with RSV-NCP by Pull-Down Assay
5.1 Materials and Methods
5.1.1 Pull-down assay to verify the interaction between VAP-B and RSV-NCP
5.2 Results
5.2.1 Vector construction
5.2.2 Pull-down assay
5.3 Discussion
CHAPTER-Ⅵ Effect of RNA interference of VAP-B on infection and relative expression of RSV in SBPH
6.1 Materials and Methods
6.1.1 ds RNA synthesis
6.1.2 Microinjection of the ds RNA to the SBPH
6.1.3 Effect of RNAi of VAP-B on infection and relative expression of RSV
6.1.4 Effect of RNAi of VAP-B on the transmission of RSV
6.2 Results
6.2.1 ds RNA construction
6.2.2 Effect of ds VAP-B injection on the expression of VAP-B in the SBPH
6.2.3 Effect of RNAi of VAP-B on infection and relative expression of RSV in the viruliferous SBPH
6.2.4 Effect of RNAi of VAP-B on the transmission of RSV
6.3 Discussion
CHAPTER-Ⅶ First reports of Tomato chlorosis virus and Potato leafroll virus in tomato from Pakistan
7.1 Materials and Methods
7.1.1 Samples collection
7.1.2 ELISA
7.1.3 RNA isolation and RT-PCR
7.1.4 Whitefly transmission
7.1.5 Next-Generation Sequencing
7.1.6 BLAST and phylogenetic analysis
7.1.7 Selection pressure and recombination analysis
7.2 Results
7.2.1 ELISA for different viruses
7.2.2 RT-PCR
7.2.3 Transmission test
7.2.4 Sequencing,BLAST and Phylogenetic analysis
7.2.5 Full genome sequence analysis
7.2.6 Selection pressure and recombination analysis
7.2.7 Gene flow and genetic differentiation analysis
7.2.8 Haplotype and nucleotide diversity
7.3 Discussion
CHAPTER-Ⅷ Summary
Bibliography
Acknowledgement
Author's Biography
Appendix Ⅰ
Appendix Ⅱ
本文編號：4016595
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